THE GIEMSA-STAINING CENTROMERES OF NIGELLA DAMASCENA

Transcription

1 J. Cell Sci. 18, 19-25(1975) 29 Printed in Great Britain THE GIEMSA-STAINING CENTROMERES OF NIGELLA DAMASCENA G.E.MARKS John Innes Institute, Colney Lane, Norwich, NR4 7 UH, England SUMMARY The centromere regions of each chromosome in the complement of Nigella damascena {zn = 2X = 12) stain differentially with Giemsa at interphase and throughout all the principal stages of mitosis and meiosis. Each centromere is seen to consist of a pair of sister half-centromeres which appear as 2 differentially stained dots. The appearance and behaviour of these dots indicates that they are kinetochores. The technique used does not stain centromeres in other plant species investigated, a fact which shows that the centromeres of Nigella are in some way different. The implications of this observation in relation to centromere polymorphism are discussed. INTRODUCTION Chromosome preparations of Nigella damascena (zn = zx =12) stained with Giemsa show differential staining at the centromeres of all the chromosomes in the complement. This phenomenon has been studied at all the principal stages of mitosis and the results are presented in this paper. METHODS Root tips and anthers were collected from plants of Nigella damascena grown from commercial seed of unknown origin. Giemsa preparations were made as follows. Root-tips 1. Fresh or pretreated (005 % aqueous colchicine for 4 h at room temperature) root tips were fixed in 3:1 ethanol/acctic acid overnight. 2. Root tips were then transferred to 45 % acetic acid (v/v) at 60 C, incubated for 20 min, and then transferred to distilled water at room temperature. This treatment softened the root tips, allowing satisfactory squashing and also proved to be the most critical and essential step in obtaining good differentiation with Giemsa. 3. Squash preparations were made in 45 % acetic acid at room temperature on clean slides without heating. There was no subsequent loss of cells if the squashing and spreading were satisfactory; this was checked under phase-contrast. Coverslips were removed using the dryice method and the slides air-dried on a warm hotplate. 4. Slides were immersed in a fresh saturated solution of barium hydroxide at room temperature for 5-10 min, then rinsed in distilled water and washed in tap water for 1 h. 5. The slides were then incubated in twice strength SSC (salt-sodium citrate) at 60 C for 1 h. 6. After rinsing in distilled water the slides were put into 2% Giemsa (G. T. Gurr's R66 improved stock diluted x 50 with M/15 SOrensen's phosphate buffer, ph 69). Staining was checked after 5 min and thereafter at intervals until the preparation was optimally stained, which usually occurred within 1 h.

2 20 G. E. Marks 7. Slides were then rinsed rapidly in distilled water, dried on a hotplate for 12 h, rinsed in Euparal Essence and mounted in Euparal. Pollen mother cells 1. Anthers were fixed in Pienar's fixative (6:3:2, v/v methanol:chloroform:propionic acid) for 24 h and stored for 1 week in 90% v/v ethanol at +4 C. Storage in ethanol prevents disintegration of pollen mother cells during squashing. 2. Squash preparations were made in a drop of 45 % acetic acid and observed under phasecontrast. Slides containing suitable division stages were heated very slightly after gently pressing the coverslip. No adhesive was necessary because it was found that if squashing and spreading were satisfactory there was negligible loss of cells. 3. Coverslips were removed using dry ice and the slides air-dried on a warm hotplate. 4. Slides were then incubated in 45 % acetic acid at 60 C for 20 min and washed for 15 min in tap-water, finally rinsing in distilled water. Thereafter slides were treated as from stage 4 given above for root tip preparations. Preparations of root tips and pollen mother cells were also made using conventional stains (carmine, Feulgen, orcein, toluidine blue) for comparative purposes. RESULTS The chromosome complement of Nigella damascena consists of 5 pairs of metacentric chromosomes and one pair of acrocentric chromosomes. With conventional chromosome stains the centromeres appear as the usual featureless unstained gaps. However with Giemsa staining this gap contains one pair of darkly stained dots which, together with one pair of telomeres, are the only regions throughout the chromosome complement which stain differentially with Giemsa (Fig. 1). At interphase, Giemsa staining shows chromocentres (Fig. 2). In a sample of 100 cells the mean number found was ± At all the principal stages of mitosis the centromere is differentially stained. At prophase (Fig. 3), prometaphase (Fig. 4) and metaphase (Fig. 5), there is a pair of dots at each centromere, whereas at anaphase each centromere shows a single dot (Fig. 6). At all stages of meiosis the centromere is likewise differentially stained with Giemsa. It is clearly seen at pachytene (Fig. 7) and its doubleness is evident at metaphase I (Fig. 8), anaphase I (Fig. 9) and metaphase/anaphase II (Figs. 10, 11). It appears as a single dot at anaphase II (Fig. 12). DISCUSSION The term centromere is used here simply to describe that region of the chromosome concerned with chromosome attachment and movement on the spindle. Essentially the results show that at the centromere of each chromosome in Nigella there is a Giemsastaining body bridging the space across each chromatid arm. Chromosomes therefore have a pair of dots and chromatids a single one. At metaphase of mitosis the pairs of dots are clearly orientated parallel to the spindle axis in an amphitelic manner (Fig. 5), whereas at metaphase I of meiosis the pairs are co-oriented syntelically (Fig. 8). Such orientation in mitosis and meiosis is what we expect of centromeres from the study of chromosome orientation and movement in Tipula oleracea (Bauer, Dietz & Robbeln, 1961). At anaphase, both in mitosis and meiosis, it is the stained dots which lead the way to the spindle poles (Figs. 6, 9).

3 Nigella centromeres 21 From the foregoing evidence there seems little doubt that what stains differentially with Giemsa is some structure within the confines of the centromere. Lima-de-Faria (1956) has described structures at the centromeres of chromosomes in many species, including both animals and plants, and has postulated a generalized structure for the centromere. One part of this structure contained chromomeres which were thought to be concerned with spindle attachment, that is, they were 'kinetochores'. Stack (1974) considers the centromeric dots which he describes in Allium, Ornithogalum, Rhoeo and Tradescantia chromosomes to be kinetochores and claims that the Giemsa technique employed is specific for demonstrating them. Likewise, similar pairs of dots at the centromeres of human metaphase chromosomes have been demonstrated by Eiberg (1974) who considers these also to be the result of using a specific Giemsa method. Evans & Ross (1974) equated these pairs of dots in human chromosome centromeres with the pairs of unstained spheres often seen in the centromeres of human, mouse and other mammalian preparations pretreated with colcemid and stained conventionally with orcein or Giemsa. They also believe that the pair of raised prominences seen at the centromere regions in optically shadowed unstained human chromosomes pretreated for either C or G banding to be equivalent to the dots. Since it is known from ultrastructural studies that the regions occupied by the raised prominences contain paired disk-shaped kinetochores (Jokelainen, 1967) it is suggested by Evans & Ross (1974) that the structures seen with light microscopy are indeed the kinetochores. If their conclusion is correct and it applies equally to the paired dots in Nigella chromosomes we can expect these also to have demonstrable kinetochores under the electron microscope. Investigations of this possibility are now in progress. Centrometric chromomeres are known in some cases to contain DNA (Lima-de- Faria 1950, 1956). The Nigella centromere, however, does not stain with Feulgen, neither does it show allocycly with respect to Giemsa staining. Such allocycly is, however, evident in the Giemsa-stained centromeres described by Stack (1974), for in the examples given the centromeres are visible only during division and they do not produce chromocentres at interphase. The Giemsa method described here is not specific for staining centromeres because it fails to stain them in other species. In fact it stains telomeres only in Clematis montana, pericentric heterochromatin in Rhoeo discolor and it differentiates intercalary bands only in the chromosomes of various Anemone species (Marks & Schweizer, 1974, and G. E. Marks, unpublished). If the technique has any specificity, therefore, it must be for a particular chromosome component which is common to all those chromosome regions which it stains. Telomeres sometimes show neo-centric activity as in Secale and Zea (Prakken & Muntzing, 1942; Rhoades, 1952), and they may take over the role of the centromere, as in scorpion chromosomes (Piza, 1974). However, these indications of functional similarities between centromeres and telomeres are not paralleled by the abilities of these regions to stain differentially with Giemsa, because the pair of differentially stained telomeres in Nigella do not show neo-centric activity (Fig. 6). Because centromeres from different species react differently to the same technique we must conclude that centromeres are not necessarily similar even in the chromo-

4 22 G. E. Marks somes of species within related genera (e.g. Anemone, Clematis and Nigella). If we accept the concept of centromere polymorphism then caution is needed in formulating generalizations about centromere structure from scattered observations made on a range of organisms. I wish to acknowledge the helpful discussions of the work presented in this paper with Professors D. R. Davies and L. F. La Cour, F.R.S., and Dr D. Schweizer. REFERENCES BAUER, H., DIETZ, R., & ROBBELN, C. (1961). Die Spermatocytenteilungen der Tipuliden. Ill Mitteilung. Das Bewegungsverhalten der Chromosomen in Translokationsheterozygaten von Tipula oleracea. Chromosoma 12, EIBERG, H. (1974). New selective Giemsa techniques for human chromosomes, Cd staining. Nature, Lond. 248, 55. EVANS, H. J. & Ross, A. (1974). Spotted centromeres in human chromosomes. Nature, Lond. 249, 861. JOKELAINEN, P. T. (1967). The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J. Ultrastruct. Res. 19, LIMA-DE-FAHIA, A. (1950). The Feulgen test applied to centromeric chromomeres. Hereditas 36, LIMA-DE-FARIA, A. (1956). The role of the kinetochore in chromosome organisation. Hereditas 42, MARKS, G. E. & SCHWEIZER, D. (1974). Giemsa banding: karyotype differences in some species of Anemone and in Hepatica nobilis. Chromosoma 44, PIZA, S. DE T. (1974). Scorpion chromosomes with centromeres at both ends. In Chromosomes Today, vol. 4 (ed. J. Wahtman & K. Lewis), p New York and Jerusalem: John Wiley & Sons Ltd. and Israel University Press. PRAKKEN, R. & MUNTZING, A. (1942). A meiotic peculiarity in rye, simulating a terminal centromere. Hereditas 28, RHOADES, M. M. (1952). Preferential segregation in maize. In Heterosii, pp Ames: Iowa State College Press. STACK, S. M. (1974). Differential Giemsa staining of kinetochores and nucleolus organizer heterochromatin in mitotic chromosomes of higher plants. Chromosoma 47, (Received 13 November 1974) Figs Microphotographs of mitosis in Nigella damascena. Fig. 1 is from a colchicine-treated root tip. Figs. 2-6 are from untreated material. All preparations are Giemsa stained, x Fig. 1. Colchicine metaphase showing full complement of 5 pairs of metacentric and 1 pair of acrocentric chromosomes (ac). All chromosomes have stained centromeres and one pair of metacentrics show faintly stained telomeres (arrows). Fig. 2. Interphase nucleus showing 12 distinct and 4 indistinct chromocentres. The former most probably correspond to the centromeres and the latter probably represent 2 pairs of sister telomeres. Fig. 3. Prophase. Centromeres consist of a pair of stained dots (sister half-centromeres). Fig. 4. Prometaphase. Repulsion between sister half-centromeres is evident in most chromosomes. Fig. 5. Metaphase. Amphitelic orientation of sister half-centromeres is clear in all chromosomes. Fig. 6. Anaphase. Half centromeres lead the way on the spindle and are clustered towards the poles. The stained telomeres (arrows) do not show neo-centric behaviour.

5 Nigella centromeres f ac 4 10 //m, ft

6 24 G. E. Marks Figs Microphotographs of meiosis in Nigella damascena. Giemsa. X Fig. 7. Pachytene. Pairing is complete and all centromere regions and one telomere region are differentially stained. The stained telomere is strictly terminal (arrow) whereas the centromere of the acrocentric bivalent is subterminal (ac). Fig. 8. Metaphase I. Six bivalents all showing distinct differentially stained centromeres. Some of the centromeres are clearly double structures in which the syntelic orientation of the sister half-centromeres is evident. Fig. 9. Anaphase I. The doubleness of each centromere is evident in most chromosomes. Fig. 10. Metaphase II. Polar view showing the centromeres located at the periphery of the spindle. Fig. 11. Metaphase/anaphase II. Slightly oblique polar view showing the amphitelic orientation of sister half-centromeres. Fig. 12. Anaphase II. Spread out pair of groups showing a single half-centromere located to one side of each chromatid.

7 Ntgella centromeres 10 /mi Ji * m * J 12 1

8